Porous orthopedic materials coated with demineralized bone matrix

ABSTRACT

A biomaterial including a porous biocompatible structure having interconnected pores, wherein the pores have interior walls and are interconnected by passageways, the interior walls and passageways being coated with an osteoinductive aqueous demineralized bone extract solution, the aqueous demineralized bone extract solution including growth factors, proteins, a demineralized bone matrix and at least one of a weak acid and a guanidine hydrochloride, wherein the demineralized bone matrix is present per 100 g of the solution in an amount of from about 2 g to about 10 g.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/688,912 filed Mar. 21, 2007, and entitled “Porous OrthopedicMaterials Coated with Demineralized Bone Matrix,” the disclosure ofwhich is expressly incorporated in its entirety herein by thisreference.

TECHNICAL FIELD

This invention relates generally to coated porous orthopedic materialsand more specifically to porous orthopedic materials coated withdemineralized bone matrix extracts comprising growth factors.

BACKGROUND OF THE INVENTION

Prosthetic devices and bone implants can either be made of resorbable ornon-resorbable materials. In particular, current bone graft materialsinclude autografts (bone material obtained from the patient), allografts(cadaver bone and bone material typically obtained from tissue banks);xenografts (bone materials from animals), and a variety of artificial orsynthetic bone substitute materials. Such bone substitute materialsinclude materials that are biocompatible with existing bone, tendon,cartilage and ligaments, and may comprise metals, ceramics, or compositematerials. Although synthetic materials can be designed to have porousstructures that can accommodate de-novo bone in-growth, they aregenerally considered inadequate as being non-osteoinductive.

The prior art has extracted growth factors from demineralized bonematrix (DBM) to be used as a surface coating or a putty to induce bonegrowth into implant materials. Demineralized bone matrix (DBM) is a wellcharacterized osteoinductive resorbable material containing growthfactors, osteogenic proteins and collagen, which has also been extractedfrom DBM to be used as a gel coating for implant materials, U.S. PatentApplication No. 2003/0044445 discloses a DBM soluble extract of proteinsthat is dried, reconstituted and then mixed with demineralized boneparticles to provide a bone filling material. However, there is noteaching of applying a DBM soluble extract to a porous synthetic implantmaterial where the extract coats within the pores.

Alternatively, U.S. Pat. No. 6,576,249 discloses a method for preparinga bone gel and bone putty by dissolving DBM in water, allowing it toform a gel and mixing it with non-demineralized bone particles to formputty. The '249 patent does not disclose using this material as acoating. The presence of the bone particles in the material wouldprohibit it from coating the pores of a porous implant material.

U.S. Pat. No. 6,376,573 discloses a porous ceramic implant material ofcoralline hydroxyapatite having a coating within the pores of thematerial. The coating however, is used to reinforce the implant materialand not to promote bone growth. The coating therefore cannot fill thepores, but must only be on the walls of the pores. This is accomplishedby adding the coating as a liquid and then catalyzing the conversion toa polymeric material in situ.

As can be seen, there is a need for a coating material for porousimplants that promotes bone growth, allowing the integration of theimplant within the patient. It would be desirable for the coating tocomprise growth factors, including osteoinductivc proteins to promotethe bone growth.

SUMMARY OF THE INVENTION

In one aspect of the present invention there is provided a biomaterialcomprising a porous biocompatible structure comprising interconnectedpores, wherein the pores comprise interior walls and may beinterconnected by passageways, an aqueous demineralized bone extractcoating comprising growth factors, proteins or a combination thereof, ademineralized bone gelatin coating comprising a demineralized bonematrix gelatin, and wherein the demineralized bone extract coating andthe demineralized bone gelatin coating may cover the interior walls andpassageways. The demineralized bone extract coating and thedemineralized bone gelatin coating may be combined into a single coatingbefore being applied to the porous biocompatible substrate.Alternatively, the demineralized bone extract coating may be applied tothe porous biocompatible substrate and then the demineralized bonegelatin coating may be applied over the demineralized bone extractcoating.

In another aspect of the present invention, there is provided abiomaterial comprising a porous biocompatible structure comprisinginterconnected pores, wherein the pores comprise interior walls and areinterconnected by passageways and an aqueous demineralized bone extractcoating comprising growth factors, proteins or a combination thereofwherein the demineralized bone extract coating covers the interior wallsand passageways.

In a further aspect of the present invention, there is provided anorthopedic implant comprising a porous biocompatible structurecomprising; interconnected pores, wherein the pores comprise interiorwails and are interconnected by passageways and wherein the pores havean average diameter of from about 200 microns to about 500 microns andan aqueous demineralized bone extract coating comprising growth factors,proteins or a combination thereof, wherein the demineralized boneextract coating covers the interior walls and passageways, wherein thebone extract coating is dried on the interior walls and passageways.

In yet another aspect of the present invention there is provided amethod of preparing a biomaterial comprising mixing demineralized bonewith an aqueous solution, the aqueous solution comprising a weak acid orguanidine hydrochloride, and wherein the mixing proceeds with constantagitation at a temperature of no greater than 50° C. for a time periodof from about 8 hours to about 96 hours to prepare a demineralized boneextract; separating the demineralized bone extract from any remainingsolids; diluting, removing or neutralizing the weak acid or guanidinehydrochloride in the demineralized bone extract; coating a porousbiocompatible structure with the demineralized bone extract, wherein theporous biocompatible structure has a porosity comprising interconnectedpores, the pores comprising interior walls and interconnected bypassageways, and wherein the demineralized bone extract infiltrates thepores and coats the interior walls and passageways; and drying theapplied demineralized bone extract onto the porous biocompatiblestructure. The method may further comprise the steps of mixing thesolids separated from the extract with an aqueous saline solution toform a suspension; heating the suspension to a temperature of from about85° C. to about 130° C. at a pressure of at least 15 psig, dissolvingthe demineralized bone to produce a demineralized bone gelatin; andmixing the demineralized bone gelatin with the demineralized boneextract before coating the biocompatible structure. Alternatively, themethod may further comprise the steps of mixing the solids separatedfrom the extract with an aqueous saline solution to form a suspension;heating the suspension to a temperature of from about 85° C. to about130° C. at a pressure of at least 15 psig, dissolving the demineralizedbone to produce a demineralized bone gelatin solution; applying thedemineralized bone gelatin solution over the dried demineralized boneextract on the biocompatible structure, wherein the demineralized bonegelatin solution infiltrates the pores and coats the internal walls andpassageways; allowing the applied demineralized bone gelatin solution togel; and lyophilizing the biocompatible structure and the applieddemineralized bone gelatin solution.

These and other features, aspects and advantages of the presentinvention will become better understood with reference to the followingdescription and claims.

DETAILED DESCRIPTION

The following detailed description is of the best currently contemplatedmodes of carrying out the invention. The description is not to be takenin a limiting sense, but is made merely for the purpose of illustratingthe general principles of the invention, since the scope of theinvention is best defined by the appended claims.

Broadly, the present invention provides a biomaterial comprising aporous biocompatible structure that may be coated with a demineralizedbone (DBM) extract comprising growth factors that increase de novo bonegrowth into the porous composite material. Methods are also provided formaking the biomaterial of the present invention. The biomaterial mayfurther comprise a demineralized bone gelatin coating over the extractcoating or the demineralized bone extract may be combined, with thegelatin coating. The biomaterial is coated such that the pores and anyconnecting passageway's are coated to support, but not block, bonegrowth into the biomaterial. The biomaterial may be in a form such as,but not limited to, granules, blocks, cylinders or pre-formed shapessuch as hip or knee augments, hip or knee implants, or other orthopedicdevices.

The biomaterial of the present invention may comprise a porousbiocompatible structure where the porous biocompatible structurecomprises pores interconnected by passageways. The porous biocompatiblestructure may be coated with an osteoinductive coating such that thecoating is not only on the surface but may also coat the pores andpassageways. It has been discovered that by coating the pores and thepassageways, bone growth into the porous material may be scatteredthroughout most of the implant compared to the porous biomaterial aloneor the porous biomaterial with just a gelatin coating. In contrast tothe present invention, coatings of the prior art coat the outer surfaceof a biomaterial with a gelatin coating and are not designed to be usedsolely on a porous substrate. Many of the coatings of the prior artcomprise demineralized bone powder which, although finely milled,prohibits the coating from completely coating the pores and passagewaysof a porous biomaterial and may even clog the pores. Other coatings ofthe prior art have been designed to strengthen porous biomaterials andare not osteoinductive.

In one embodiment, the biomaterial of the present invention may comprisea porous biocompatible structure which may comprise interconnectedpores. The pores may comprise interior walls and may be connected bypassageways. In one illustrative embodiment, the pores may have anaverage size from about 5 microns to 1000 microns or from about 200microns to about 500 microns. In another illustrative embodiment, theporous biocompatible structure may be resorbable as new bone is formedor may be non-resorbable. The resorbable biocompatible structure may be,but not limited to, hydroxyapatite. The hydroxyapatite may be tricalciumphosphate such as, but not limited. to, Calcigen® PSI. Alternatively,the hydroxyapatite may he a coralline hydroxyapatite such as, but notlimited to, the coralline hydroxyapatite described in U.S. Pat. No.4,976,736 (herein incorporated by reference), known under the trade namePro Osteon™. By way of non-limiting example, the corallinshydroxyapatite may be Pro Osteon™ 200 or Pro Osteon™500 which may havean average pore size of 200 microns and 500 microns, respectively. In analternate illustrative embodiment, the resorbable biocompatiblestructure may be, but not limited to, xenograft demineralized cancellousbone. The demineralized cancellous bone may be formed from cancellousbone from an animal such as, but not limited to, pig, cow or horse. Thecancellous bone may be cleaned to remove all blood and marrow from thepores. The bone is then demineralized in hydrochloric acid or any otherprocedure known in the art. Following the demineralization, the bone maybe washed and extracted guanidine hydrochloride. The remaining porouscollagen structure that may have the architecture of cancellous bone maythen be used in the present invention. In a further illustrativeembodiment the porous biocompatible structure may be non-resorbable. Byway of a non-limiting example, the non-resorbable biocompatiblestructure may comprise porous metal where the metal may be stainlesssteel, titanium, titanium alloy, tantalum or cobalt-chromium alloy.Examples of porous metal for use as implant material may be found inU.S. Pat. Nos. 6,206,924, 7,597,715 and 8,292,967, all of which areincorporated herein by reference. The porous biocompatible structure mayhave a form such as, but not limited to, granules, blocks, cylinders orpre-formed shapes such as hip or knee augments, hip or knee implants, orother orthopedic devices.

The biomaterial may further comprise an aqueous demineralized boneextract coating comprising growth factors, proteins or a combinationthereof. It will be appreciated that many growth factors may be proteinsand the two categories are not mutually exclusive, one is not a subsetof the other. The growth factors or proteins may be osteoinductive,helping to promote de novo growth of new bone. Non-limiting examples ofgrowth factors may be bone morphogenic proteins, particularly BMP-2 andBMP-7, TGF-β, IGF-1, VEGF, PDGF, FGF, EGF or mixtures thereof. Theaqueous demineralized bone extract coating may also comprise biologicsolutions such as, but not limited to, blood, platelet rich plasma,platelet poor plasma concentrated plasma, bone marrow aspirate,concentrated bone marrow aspirate or combinations thereof.

In one illustrative embodiment, the aqueous demineralized bone extractmay be an acid soluble demineralized bone extract. The acid solubleextract may be produced by mixing demineralized bone matrix (DBM) with aweak acid such as, but not limited to citric acid, acetic acid, lacticacid, malic acid or mixtures thereof. The mixture may be stirred oragitated from about 8 hours to about 96 hours at a temperature notgreater than 50° C. At temperatures greater than 50° C. there may beinactivation of the growth factors and/or proteins. The extract may thenbe filtered to remove any remaining solids, the acid neutralized orremoved and used to coat the porous biocompatible structure.

In another illustrative embodiment, the aqueous demineralized boneextract may be a guanidine hydrochloride demineralized bone extract. Theguanidine hydrochloride demineralized bone extract may be produced bymixing DBM with a solution of guanidine hydrochloride where theguanidine hydrochloride may be from about 3M to about 6M. The mixturemay be stirred or agitated from about 8 hours to about 96 hours at atemperature not greater than 50° C. The extract may then be filtered toremove any remaining solids, the guanidine hydrochloride neutralized orremoved and used to coat the porous biocompatible structure. Althoughnot wishing to be bound by theory, the guanidine hydrochloridedemineralized bone extract may have a higher protein content than theacid soluble demineralized bone extract as it may be more likely todissolve some of the DBM.

The aqueous demineralized bone extract coating may be applied to theporous biocompatible structure in a manner that allows for the coatingto be delivered to the pores and passageways. For example, the coatingmay be applied under vacuum.

Alternatively, the coating may be applied by placing the material in theextract and allowing the extract to enter the pores and passageways bycapillary action. Once the coating is applied to the biocompatiblestructure it may be dried onto the structure.

In another embodiment the biomaterial of the present invention mayfurther comprise a demineralized bone gelatin coating. The demineralizedbone gelatin coating may be formed from the remaining solids after theextract is filtered or it may be formed from a different demineralizedbone sample. Alternatively, it may be formed from partially purified orisolated collagen. The demineralized bone or collagen may be mixed witha saline solution, water or any other biocompatible solution and heatedunder pressure to dissolve the demineralized bone matrix or collagen toform the demineralized bone gelatin coating. The coating may then becoated over the demineralized bone extract coating or it may be mixedwith the demineralized bone extract coating and the mixture of theextract and gelatin coatings may then be applied to the biocompatiblestructure to give only a single coating. The demineralized gelatincoating is applied such that it coats the pores and the passageways. Aswith the demineralized bone extract coating, the gelatin coating, eitheralone or combined with the extract coating, may be applied under vacuumor it may coat the pores through capillary action. After application tothe biocompatible structure, the demineralized bone gelatin coating maybe dried. The demineralized bone gelatin coating may be less viscous athigher temperatures making it easier to apply to the biocompatiblestructure. However, care should be taken so that the demineralized bonegelatin coating is not at a temperature high enough to inactivate thegrowth factors and/or proteins of the demineralized bone extractcoating.

It will be appreciated that although the embodiments describe a singlecoating, more than one coating of either the extract and/or the gelatinmay be applied. If more than one coat is applied, the individual coatsmay be dried before the next coat is applied.

In one embodiment, the present invention provides a method of preparinga biomaterial comprising mixing DBM with an aqueous solution of a weakacid or guanidine hydrochloride with constant agitation for a set amountof time to produce an aqueous demineralized bone extract, filtering theextract solution to remove any remaining solids, neutralizing orremoving the weak acid or guanidine hydrochloride and coating the porousbiocompatible structure with the extract. The amount of DBM may be fromabout 2 g to about 10 g per 100 g of solution. The DBM may be in anyform, including, but not limited to, powder, granules, fragments,slices, pellets, slices or shavings. It will be appreciated that theconcentration of growth factors and/or proteins in the extract may berelated to both the amount of DBM used and the form, as well as thestrength of aqueous solution. The aqueous solution may comprise anybiologically compatible aqueous solution, particularly those in whichgrowth factors and proteins may be stable in. Examples of such solutionsmay be, but not limited to, Tris buffer, Tris buffered saline, phosphatebuffer and phosphate buffered saline. In one illustrative embodiment,the solution may be a weak acid solution where the weak acid may be, butnot limited to, citric, acid, lactic acid, malic acid, ascorbic acid orcombinations thereof. Any weak acid known in the art may be used. Theconcentration of the weak acid solution may be from about 2 M to about 3M. In a second illustrative embodiment, the solution is a guanidinehydrochloride solution where the concentration of the guanidinehydrochloride solution may be from about 3 M to about 6 M.

The DBM may be mixed with the aqueous solution for a set amount of timewith constant agitation at a temperature not greater than 50° C. Theamount of time that the DBM and aqueous solution may be mixed may befrom about 8 hours to about 96 hours. In one illustrative embodiment,the DBM and aqueous solution may be mixed together from about 24 hoursto about 96 hours. The DBM and aqueous solution may be mixed togetherwith constant agitation during that time. Constant agitation may beobtained by, but not limited to, stirring, shaking, ultrasound or anycombination thereof as well as any other methods of agitating a mixture.The mixing may be carried out at a temperature that may be conducive toextracting growth factors and/or proteins from the DBM, but where growthfactors and/or proteins may be stable. In an illustrative embodiment,the temperature may be no greater than 50° C. In another illustrativeembodiment, the temperature may be room temperature.

After mixing for the appropriate amount of time, the resultingdemineralized bone extract may be separated from any insoluble DBMremaining. This separation may occur by any number of processes such as,but not limited to, decanting, filtering or centrifuging. In oneillustrative embodiment, the solution may be filtered to remove anysoluble DBM remaining. The size of the sieve or filter will depend onthe size of the DBM particles remaining, which may further depend on theinitial form of DBM. In one illustrative embodiment, the filter may befrom about 50 microns to about 300 microns. The filter may be a sieve,paper, scintered glass, woven or non-woven fabric, or any other means offiltering that is known in the art.

The demineralized bone extract may be diluted, neutralized or the weakacid or guanidine hydrochloride removed. Methods for doing this may be,but not limited to, titration, dialysis, liquid-liquid extraction,hollow fiber filtration, ultrafiltration, crossflow filtration orprecipitation. In one illustrative embodiment aqueous solution may beneutralized to a pH of from about 6.5 to about 7.5 by titration with anappropriate counterion. Such methods are well known in the art. Inanother illustrative embodiment, the weak acid or guanidinehydrochloride may be removed by dialysis, hollow fiber filtration,ultrafiltration or crossflow filtration against a biologicallycompatible buffer, such as, but not limited to, Tris, IBS, phosphate,PBS or water, where the pH of the buffer may be from about 6.5 to about7.5. The molecular weight cutoff of the dialysis membrane will depend onthe size of the proteins and/or growth factors desired in the solution.The dialysis, hollow fiber filtration, ultrafiltration or crossflowfiltration membrane may have, for example, a molecular weight cut offless than or equal to 12 Kd or from about 10 Kd to about 12 Kd. It iswell known in the art how to select the molecular weight cut off ofdialysis tubing to retain the desired molecules within the sample.

Once neutralized, a porous biocompatible structure may be coated withthe demineralized bone extract. The demineralized bone extract may beapplied to the biocompatible structure such that the demineralized boneextract infiltrates the pores and passageways of the biocompatiblestructure. In one illustrative embodiment, the demineralized boneextract may be applied to the biocompatible structure under vacuum. Inanother illustrative embodiment the demineralized bone extract may beapplied to the biocompatible structure by dipping the structure into theextract and allowing it to infiltrate the pores and passageways bycapillary action. After the demineralized bone extract has been appliedto the biocompatible structure, it may be dried onto the biocompatiblestructure. The demineralized bone extract may be dried onto thestructure by lyophilization, vacuum, heating at a temperature notgreater than 50° C. or a combination thereof.

In another embodiment, the method of the present invention may furthercomprise making a demineralized bone gelatin. The demineralized bonegelatin may be coated over the demineralized bone extract or it may bemixed with the extract before coating to form a single coating. Thedemineralized bone gelatin may be formed by mixing DBM with an aqueoussaline solution such as, but not limited to PBS, TBS or a sodiumchloride solution, to form a suspension. The suspension may be treatedto increased temperature and pressure such as, but not limited to,autoclaved. In one illustrative embodiment, the solution may be heatedto a temperature of from about 85° C. to about 130° C. at a pressure ofat least about 15 psig. The DBM may be dissolved to produce ademineralized bone gelatin. Methods for forming a demineralized bonegelatin are known in the art. The DBM may be the solids removed duringthe filtering step while forming the demineralized bone extract or itmay be fresh DBM. Alternatively, it will be appreciated that since thedemineralized bone gelatin comprises mainly collagen, collagen of anypurity may be substituted for the DBM.

The demineralized bone gelatin may be coated over the drieddemineralized bone extract coating on the biocompatible structure suchthat the gelatin coats the pores and passageways. Alternatively, thedemineralized bone gelatin may be mixed with the demineralized boneextract prior to the extract being coated onto the biocompatiblestructure to form a single coating. The single coating comprising thedemineralized bone extract and gelatin may then be applied to thebiocompatible structure such that the pores and passageways are coated.It will be appreciated that the demineralized bone gelatin may be lessviscous at higher temperatures, making it easier to apply to thebiocompatible structure. If the demineralized bone gelatin is applied tothe biocompatible structure in a less viscous form such as a solution,it should be allowed to gel before any other steps are performed. Careshould be taken so that the demineralized bone gelatin coating is not ata temperature high enough to inactivate the growth factors and/orproteins of the demineralized bone extract coating.

Once applied, the demineralized bone gelatin, either alone or mixed withthe demineralized bone extract, may be dried onto the biocompatiblestructure. The demineralized bone gelatin may be dried onto thestructure by lyophilization, vacuum, heating at a temperature notgreater than 50° C. or a combination thereof.

It should be understood, of course, that the foregoing relates toexemplary embodiments of the invention and that modifications may bemade without departing from the spirit and scope of the invention as setforth in the following claims.

What is claimed is:
 1. A biomaterial comprising: a porous biocompatiblestructure comprising interconnected pores, wherein the pores compriseinterior walls and are interconnected by passageways; an aqueousdemineralized bone extract coating comprising growth factors, proteinsor a combination thereof; and a demineralized bone gelatin coatingcomprising a demineralized bone matrix gelatin; wherein thedemineralized hone extract coating and the demineralized bone gelatincoating cover the interior walls and passageways.